Adenovirus-mediated Expression of a Truncated PDGFβ Receptor Inhibits Thrombosis and Neointima Formation in an Avian Arterial Injury Model

2001 ◽  
Vol 86 (09) ◽  
pp. 914-922 ◽  
Author(s):  
Hongliu Ding ◽  
Rongqing Wang ◽  
Robin Marcel ◽  
Daniel Fisher
Keyword(s):  
Growth Factor ◽  
Recombinant Adenovirus ◽  
Thrombus Formation ◽  
Arterial Injury ◽  
Neointima Formation ◽  
Injury Model ◽  
Powerful Approach ◽  
And Migration

SummaryPlatelet-derived growth factor (PDGF) is a major mediator of neointima formation after arterial injury. We constructed a recombinant adenovirus, Ad/PDGFtr, that expresses the soluble extracellular domain of the murine PDGFβ receptor (PDGFtr). The expressed PDGFtr was appropriately glycosylated and secreted by chicken vascular smooth muscle cells (SMCs) in vitro. The expressed PDGFtr inhibited human PDGF-BB induced receptor autophosphorylation, and also inhibited PDGF-BB induced cell proliferation without affecting PDGF-AA induced mitogenesis. In vivo transduction of balloon-injured rooster femoral arteries with Ad/PDGFtr resulted in expression and secretion of the glycosylated PDGFtr. The expressed PDGFtr significantly inhibited neointima formation compared with controls. Neointima-associated thrombus was significantly reduced in Ad/PDGFtr transduced arteries compared with controls. Thus, in addition to impacting on SMC proliferation and migration, PDGF-BB plays a role in thrombus formation in response to arterial injury. Growth factor inhibition by localized gene delivery constitutes a powerful approach to intervene in the molecular pathways involved in vascular disease.

scholarly journals Inhibitory Effect of a Glutamine Antagonist on Proliferation and Migration of VSMCs via Simultaneous Attenuation of Glycolysis and Oxidative Phosphorylation

2021 ◽  
Vol 22 (11) ◽  
pp. 5602
Author(s):  
Hyeon Young Park ◽  
Mi-Jin Kim ◽  
Seunghyeong Lee ◽  
Jonghwa Jin ◽  
Sungwoo Lee ◽  
...  
Keyword(s):  
Mammalian Target Of Rapamycin ◽  
Inhibitory Effect ◽  
Metabolic Reprogramming ◽  
Neointima Formation ◽  
Artery Ligation ◽  
Carotid Artery Ligation ◽  
And Migration ◽  
Proliferation And Migration

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and glutaminolysis are increased in rapidly proliferating VSMCs to support their increased energy requirements and biomass production. Thus, it is essential to develop new pharmacological tools that regulate metabolic reprogramming in VSMCs for treatment of atherosclerosis. The effects of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, have been broadly investigated in highly proliferative cells; however, it is unclear whether DON inhibits proliferation of VSMCs and neointima formation. Here, we investigated the effects of DON on neointima formation in vivo as well as proliferation and migration of VSMCs in vitro. DON simultaneously inhibited FBS- or PDGF-stimulated glycolysis and glutaminolysis as well as mammalian target of rapamycin complex I activity in growth factor-stimulated VSMCs, and thereby suppressed their proliferation and migration. Furthermore, a DON-derived prodrug, named JHU-083, significantly attenuated carotid artery ligation-induced neointima formation in mice. Our results suggest that treatment with a glutamine antagonist is a promising approach to prevent progression of atherosclerosis and restenosis.

Absence of transforming growth factor beta 1 in murine platelets reduces neointima formation without affecting arterial thrombosis

2017 ◽  
Vol 117 (09) ◽  
pp. 1782-1797 ◽  
Author(s):  
Eva Schütz ◽  
Magdalena L. Bochenek ◽  
Dennis R. Riehl ◽  
Markus Bosmann ◽  
Thomas Münzel ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Arterial Injury ◽  
Neointima Formation ◽  
Proliferating Cells ◽  
The Media

SummaryPlatelet degranulation at the site of vascular injury prevents bleeding and may affect the chronic vascular wound healing response. Transforming Growth Factor (TGF)-β1 is a major component of platelet α-granules known to accumulating in thrombi. It was our aim to determine the role of TGFβ1 released from activated platelets for neointima formation following arterial injury and thrombosis. Mice with platelet-specific deletion of TGFβ1 (Plt.TGFβ-KO) underwent carotid artery injury. Immunoassays confirmed the absence of active TGFβ1 in platelet releasates and plasma of Plt.TGFβ-KO mice. Whole blood analyses revealed similar haematological parameters, and tail cut assays excluded major bleeding defects. Platelet aggregation and the acute thrombotic response to injury in vivo did not differ between Plt.TGFβ-KO and Plt.TGFβ-WT mice. Morphometric analysis revealed that absence of TGFβ1 in platelets resulted in a significant reduction of neointima formation with lower neointima area, intima-to-media ratio, and lumen stenosis. On the other hand, the media area was enlarged in mice lacking TGFβ1 in platelets and contained increased amounts of proteases involved in latent TGFβ activation, including MMP2, MMP9 and thrombin. Significantly increased numbers of proliferating cells and cells expressing the mesenchymal markers platelet-derived growth factor receptor-β or fibroblast-specific protein-1, and the macrophage antigen F4/80, were observed in the media of Plt.TGFβ-KO mice, whereas the medial smooth muscle-actin-immuno-positive area and collagen content did not differ between genotypes. Our findings support an essential role for platelet-derived TGFβ1 for the vascular remodelling response to arterial injury, apparently independent from the role of platelets in thrombosis or haemostasis.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals FGFRL1 Promotes Ovarian Cancer Progression by Crosstalk with Hedgehog Signaling

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Haiyan Tai ◽  
Zhiyong Wu ◽  
Su’an Sun ◽  
Zhigang Zhang ◽  
Congjian Xu
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Fibroblast Growth Factor Receptor ◽  
Growth Factor Receptor ◽  
Fibroblast Growth ◽  
Hh Signaling ◽  
And Migration

Fibroblast growth factor receptor-like-1 (FGFRL1) has been identified as the fifth fibroblast growth factor receptor. So far, little is known about its biological functions, particularly in cancer development. Here, for the first time, we demonstrated the roles of FGFRL1 in ovarian carcinoma (OC). An array and existing databases were used to investigate the expression profile of FGFRL1 and the relationship between FGFRL1 expression and clinicopathological parameters. FGFRL1 was significantly upregulated in OC patients, and high FGFRL1 expression was correlated with poor prognosis. In vitro cell proliferation, apoptosis and migration assays, and in vivo subcutaneous xenograft tumor models were used to determine the role of FGFRL1. Loss of function of FGFRL1 significantly influenced cell proliferation, apoptosis, and migration of OC cells in vitro and tumor growth in vivo. Chromatin immunoprecipitation PCR analysis and microarray hybridization were performed to uncover the mechanism. FGFRL1 expression could be induced by hypoxia through hypoxia-inducible factor 1α, which directly binds to the promoter elements of FGFRL1. FGFRL1 promoted tumor progression by crosstalk with Hedgehog (Hh) signaling. Taken together, FGFRL1 is a potential predictor and plays an important role in tumor growth and Hh signaling which could serve as potential therapeutic targets for the treatment of OC.

scholarly journals Adenovirus-Mediated Expression of a Ribozyme to c-mybmRNA Inhibits Smooth Muscle Cell Proliferation and Neointima Formation In Vivo

1999 ◽  
Vol 73 (9) ◽  
pp. 7745-7751 ◽  
Author(s):  
Dennis G. Macejak ◽  
Hua Lin ◽  
Saiphone Webb ◽  
Jennifer Chase ◽  
Kristi Jensen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Smooth Muscle ◽  
Animal Models ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Recombinant Adenovirus ◽  
Neointima Formation ◽  
Inhibition Of Proliferation

ABSTRACT Smooth muscle cell (SMC) proliferation is an important component of restenosis in response to injury after balloon angioplasty. Inhibition of proliferation in vivo can limit neointima hyperplasia in animal models of restenosis. Ribozymes against c-myb mRNA have been shown to be effective inhibitors of SMC proliferation in vitro. The effectiveness of adenovirus as a gene therapy vector in animal models of restenosis is well documented. In order to test the utility of ribozymes to inhibit SMC proliferation by a gene therapy approach, recombinant adenovirus expressing ribozymes against c-mybmRNA was generated and tested both in vitro and in vivo. This adenovirus ribozyme vector is shown to inhibit SMC proliferation in culture and neointima formation in a rat carotid artery balloon injury model of restenosis.

Empagliflozin impairs smooth muscle cell proliferation, accelerates endothelial regeneration, and prevents neointima formation by altered expression of the vasoactive peptide apelin

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
J Dutzmann ◽  
L.M Bode ◽  
L Korte ◽  
K Kalies ◽  
S Koch ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Injury ◽  
Blue Staining ◽  
Funding Source ◽  
Neointima Formation ◽  
And Migration ◽  
Vasoactive Peptide ◽  
Proliferation And Migration

Abstract Background Empagliflozin, an inhibitor of the sodium glucose co-transporter 2 (SGLT2), developed as an anti-diabetic agent exerts additional beneficial effects on heart failure outcomes in patients with type 2 diabetes mellitus at high cardiovascular risk. However, the effect of empagliflozin on vascular cell function and vascular remodeling processes remain largely elusive. Methods/Results Immunocytochemistry and immunoblotting revealed SGLT2 to be expressed in human diabetic and non-diabetic smooth muscle (SMC) and endothelial cells (EC) as well as in murine femoral arteries. In vitro, empagliflozin significantly reduced serum-induced proliferation and migration of human diabetic and non-diabetic SMCs in a dose-dependent manner without any toxic or apoptotic effects. In contrast, empagliflozin significantly increased the cell count and migrational capacity of human diabetic ECs, but not of human non-diabetic ECs. In vivo, therapeutic application of empagliflozin (225 mg/kg medicated diet) resulted in a significantly reduced number of Ki-67+ proliferating neointimal cells in response to femoral artery wire-injury in C57BL/6J mice. Empagliflozin furthermore prevented subsequent neointima formation (luminal stenosis 91.2% vs. 80.6% at 21 days; P<0.05). Comparable effects of empagliflozin were observed in a streptozocin-induced diabetic model of apolipoprotein E−/− mice. Conclusive to the in vitro-results, re-endothelialization was not significantly affected in C57BL/6 mice (non-reendothelialized distance 2.57 mm vs. 2.3 mm; P=0.07), but even significantly improved in diabetic mice after treatment with empagliflozin (3.1 mm vs. 2.58 mm; P<0.001) assessed by Evan's Blue staining 3 days after electric denudation of the murine carotid artery. Microarray analysis of human SMCs identified the vasoactive peptide apelin to be decisively regulated in response to empagliflozin treatment. Further pathway analysis exhibited apelin to prevent SMC proliferation by de-phosphorylation of Akt and to augment EC proliferation by phosphorylation of p38 MAPK. Conclusion These data document the functional impact of empagliflozin on vascular SMCs and ECs for the first time. Empagliflozin significantly reduces serum-induced proliferation and migration of SMCs in vitro and prevented neointima formation in vivo, while augmenting EC proliferation in vitro and re-endothelialization in vivo after vascular injury. Thus, empagliflozin holds promise to exert favorable effects on vascular healing, and to prevent neointima formation following vascular injury in diabetic and non-diabetic patients. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Hannover Medical School, Martin-Luther-University Halle-Wittenberg

scholarly journals Graptopetalum paraguayense Inhibits Liver Fibrosis by Blocking TGF-β Signaling In Vivo and In Vitro

2019 ◽  
Vol 20 (10) ◽  
pp. 2592 ◽  
Author(s):  
Wei-Hsiang Hsu ◽  
Se-Chun Liao ◽  
Yau-Jan Chyan ◽  
Kai-Wen Huang ◽  
Shih-Lan Hsu ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Liver Fibrosis ◽  
Liver Injury ◽  
Rat Liver ◽  
Injury Model ◽  
Ethanol Extracts ◽  
And Migration ◽  
Tgf Β1

Background and Aims: Liver fibrosis is the excessive accumulation of extracellular matrix proteins, including collagen, which occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension. Activated hepatic perivascular stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines, such as TGF-β1. The inhibition of TGF-β1 function or synthesis is a major target for the development of antifibrotic therapies. Our previous study showed that the water and ethanol extracts of Graptopetalum paraguayense (GP), a Chinese herbal medicine, can prevent dimethylnitrosamine (DMN)-induced hepatic inflammation and fibrosis in rats. Methods: We used rat hepatic stellate HSC-T6 cells and a diethylnitrosamine (DEN)-induced rat liver injury model to test the potential mechanism of GP extracts and its fraction, HH-F3. Results: We demonstrated that GP extracts and HH-F3 downregulated the expression levels of extracellular matrix (ECM) proteins and inhibited the proliferation and migration via suppression of the TGF-β1 pathway in rat hepatic stellate HSC-T6 cells. Moreover, the HH-F3 fraction decreased hepatic collagen content and reduced plasma AST, ALT, and γ-GT activities in a DEN-induced rat liver injury model, suggesting that GP/HH-F3 has hepatoprotective effects against DEN-induced liver fibrosis. Conclusion: These findings indicate that GP/HH-F3 may be a potential therapeutic agent for the treatment of liver fibrosis. The inhibition of TGF-β-mediated fibrogenesis may be a central mechanism by which GP/HH-F3 protects the liver from injury.

scholarly journals Sonic hedgehog-dependent activation of adventitial fibroblasts promotes neointima formation

2017 ◽  
Vol 113 (13) ◽  
pp. 1653-1663 ◽  
Author(s):  
Jochen Dutzmann ◽  
Alexander Koch ◽  
Simona Weisheit ◽  
Kristina Sonnenschein ◽  
Laura Korte ◽  
...  
Keyword(s):  
Sonic Hedgehog ◽  
Neointima Formation ◽  
Adventitial Fibroblasts ◽  
Initial Activation ◽  
Functional Relevance ◽  
And Migration ◽  
Activated Fibroblasts ◽  
Proliferation And Migration

Abstract Aims Adventitial cells have been suggested to contribute to neointima formation, but the functional relevance and the responsible signalling pathways are largely unknown. Sonic hedgehog (Shh) is a regulator of vasculogenesis and promotes angiogenesis in the adult. Methods and results Here we show that proliferation of vascular smooth muscle cells (SMC) after wire-induced injury in C57BL/6 mice is preceded by proliferation of adventitial fibroblasts. Simultaneously, the expression of Shh and its downstream signalling protein smoothened (SMO) were robustly increased within injured arteries. In vitro, combined stimulation with Shh and platelet-derived growth factor (PDGF)-BB strongly induced proliferation and migration of human adventitial fibroblasts. The supernatant of these activated fibroblasts contained high levels of interleukin-6 and -8 and strongly induced proliferation and migration of SMC. Inhibition of SMO selectively prevented fibroblast proliferation, cytokine release, and paracrine SMC activation. Mechanistically, we found that PDGF-BB activates protein kinase A in fibroblasts and thereby induces trafficking of SMO to the plasma membrane, where it can be activated by Shh. In vivo, SMO-inhibition significantly prevented the proliferation of adventitial fibroblasts and neointima formation following wire-induced injury. Conclusions The initial activation of adventitial fibroblasts is essential for the subsequent proliferation of SMC and neointima formation. We identified SMO-dependent Shh signalling as a specific process for the activation of adventitial fibroblasts.

Abstract 374: Inhibition of Mitochondrial CaMKII Reduces Vascular Smooth Muscle Migration and Neointima Formation and Halts Mitochondrial Mobility

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Emily Nguyen ◽  
Olha Koval ◽  
Isabella Grumbach
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Mitochondrial Fission ◽  
Leading Edge ◽  
Neointima Formation ◽  
Vsmc Proliferation ◽  
Mitochondrial Motility ◽  
And Migration

Background: Restenosis after angioplasty for coronary vascular disease remains a critical problem in cardiovascular medicine. Vascular smooth muscle cell (VSMC) migration and proliferation cause restenosis through neointima formation. Mitochondrial motility is likely necessary for cell proliferation and migration, and is inhibited in microdomains with increased Ca 2+ . The Ca 2+ /calmodulin-dependent kinase II (CaMKII) in mitochondria (mtCaMKII) is proposed to control mitochondrial matrix Ca 2+ uptake through mitochondrial Ca 2+ uniporter (MCU). Thus, we hypothesized that blocking mtCaMKII decreases VSMC migration and neointima formation by decreasing mitochondrial motility. Methods: mtCaMKII was inhibited by expression of the mitochondria-targeted CaMKII inhibitor peptide (CaMKIIN) in a novel transgenic mouse model in smooth muscle only (SM-mtCaMKIIN) or delivered by adenoviral transduction (Ad-mtCaMKIIN). Results: In our models, mtCaMKIIN was detected selectively in mitochondria of VSMC. mtCaMKIIN significantly reduced mitochondrial Ca 2+ current and Ca 2+ content compared to WT in vivo and in vitro. SM-mtCaMKIIN mice showed significantly reduced neointimal area 28 days after endothelial injury (n=8, p<0.05) and fewer proliferating neointimal cells by PCNA staining. In vitro, Ad-mtCaMKIIN mildly reduced VSMC proliferation and mitochondrial ROS production without altering maximal respiration after PDGF treatment. Ad-mtCaMKIIN abolished VSMC migration, as did mitoTEMPO and MCU inhibitor Ru360. Ad-mtCaMKIIN blocked mitochondrial mobility towards the leading edge, while relocation of mitochondria was seen in WT cells 6 h after PDGF treatment. Mitochondrial redistribution was also inhibited by Ru360, but not by mitoTEMPO or cytoplasmic CaMKII inhibition. Mitochondrial fission promotes cell migration. Accordingly, PDGF increased mitochondrial particles in WT VSMC, while mitochondria in Ad-mtCaMKIIN cells were fragmented and unresponsive to PDGF treatment. Conclusions: mtCaMKIIN prevents mitochondrial distribution to the leading edge and reduces VSMC migration and neointima formation. These data suggest mitochondrial Ca 2+ regulation plays an important role in VSMC migration by altering mitochondrial location.

Abstract 12659: Hepatoma-Derived Growth Factor Related Protein-3 as a Novel Endothelial Ligand

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Michelle LeBlanc ◽  
Weiwen Wang ◽  
Feiye Guo ◽  
Chen Shen ◽  
Rui Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Umbilical Vein ◽  
Growth Effect ◽  
Human Aorta ◽  
Related Protein ◽  
And Migration ◽  
Endothelial Ligands

Background: Endothelial ligands extrinsically regulate a broad spectrum of vascular functions with therapeutic potentials, but are traditionally identified on a case-by-case basis with technical challenges. We recently developed open reading frame phage display (OPD) for unbiased identification of phagocytosis ligands. In this study, we identified hepatoma-derived growth factor related protein-3 (HRP-3) as a putative endothelial ligand by OPD. We hypothesized that HRP-3 is a novel endothelial growth factor, capable of promoting endothelial cell (EC) growth and migration. Methods and Results: We performed 3 rounds of in vivo phage binding selection in mice with an OPD library, screened enriched phage clones by next generation DNA sequencing, and identified HRP-3 as one of the putative endothelial ligands. To confirm the finding, clonal phages displaying HRP-3, VEGF and GFP were generated and analyzed for their binding to human umbilical vein endothelial cells (HUVECs). The results show that HRP-3-Phage and VEGF-Phage had significantly higher binding to HUVECs than GFP-Phage. Functional analysis showed that purified recombinant HRP-3 significantly increased the proliferation of HUVECs at 24 and 48 h, whereas VEGF induced significant growth only at 48 h. Consistent with these findings, HRP-3 significantly stimulated cell proliferation by MTT assay. In vitro wound-healing assay indicated that both HRP-3 (500 ng/ml) and VEGF (50 ng/ml) significantly promoted the migration of HUVECs into the denuded area. To dissect the downstream signaling pathway, we demonstrated that HRP-3 significantly induced ERK1/2 phosphorylation in HUVECs after 10 min treatment. Similar effects of HRP-3 and VEGF on EC growth, migration, and ERK activation were also verified using human aorta endothelial cells. Conclusions: Our findings demonstrate that HRP-3 is a novel ligand, capable of promoting proliferation and migration of ECs. The pro-growth effect of HRP-3 is at least partially mediated through ERK pathway activation. These results in turn support the broad applicability of OPD for the systematic discovery of endothelial ligands.

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